[Value of routine preoperative oesophagogastroduodenoscopy before elective cholecystectomy]. / Stellenwert der routinemäßigen präoperativen Ösophagogastroduodenoskopie vor elektiver Cholezystektomie.

BACKGROUND: Symptomatic gallstones and inflammatory diseases of the gastroduodenum are common causes of upper abdominal pain. PATIENTS / MATERIAL AND METHODS: We evaluated the data of 766  patients who underwent pre-operative oesophagogastroduodenoscopy before elective surgical treatment of cholelithiasis between January  1, 2003 and March 31, 2008. RESULTS: Pathological findings of an inflammatory nature were seen in 43.1 % (330 / 766), 25.2 % (197 / 766) of the patients had a chronic gastritis, 14.9 % (114 / 766) an acute gastritis and 3 % (54 / 766) a gastroduodenal ulcer. Non-specific upper abdominal pain was not significantly related to an inflammatory gastroduodenal dis-ease (p = 0.0755). Independent of the history (p = 0.1065), the therapy concept had to be -changed in favour of a primary non-surgical therapy in 16.3 % (125 / 766) of the examined patients due to relevant gastroscopic findings. CONCLUSIONS: The case history is not sufficient for identifying gastroduodenal disease requiring treatment in patients with symptomatic gall-stones. Therefore, contrary to the current leading concepts, preoperative oesophagogastroduodenoscopy should be discussed as a matter of -routine in patients undergoing elective cholecystectomy in order to treat pathological gastroduodenal findings.

Cross-reactivity of anti-digoxin antibodies with digitoxin depends on tracer structure.

The most common methods for measuring digoxin concentrations in serum are immunoassays. The prerequisite for exact determination of the digoxin value is an antibody that specifically binds digoxin. Because digitoxin differs from digoxin only in the C-12 hydroxy group, it is difficult to obtain anti-digoxin antibodies that do not cross-react with this compound. During the development of a fluorescence polarization immunoassay (FPIA) for digoxin, we investigated digoxin tracers with different structures. We found that in FPIA the digitoxin cross-reactivity of an antibody could be reduced by varying the structure of the tracer molecule.

Direct analysis of polymerase chain reaction products using enzyme-linked immunosorbent assay techniques.

PCR products obtained using primers carrying at their 5' ends biotin and an antigenic group (e.g., fluorescein) can be quantitatively analyzed by immunological techniques. The procedure described here does not require electrophoretic separation and/or hybridization with radioactive probes. It takes advantage of the fact that biotinylated DNA can be immobilized on avidin- or streptavidin-coated microtiter plates and then can be quantitated by an ELISA specific for the antigenic group. The PCR/ELISA procedure is suitable for routine diagnostic purposes and lends itself to automation. The sensitivity of the immunological detection system that employs horseradish peroxidase linked to anti-fluorescein antibodies is high: 1 microliter of the PCR mixture obtained after approximately 25 cycles of amplification of 1 ng/microliter genomic template DNA is sufficient for the detection of human single-copy genes. The usefulness of the procedure for the quantitative analysis of the amount of DNA present in a blood or tissue sample is discussed.

Quantitative analysis of polymerase chain reaction (PCR) products using primers labeled with biotin and a fluorescent dye.

PCR primers covalently labeled with biotin and a fluorescent dye allow immobilization and separation of the products which can be quantitatively analyzed subsequently. The procedure we have developed circumvents electrophoretic separation and radioactive labeling. Exact quantitative analysis of reaction products is feasible during the logarithmic phase of amplification when Taq polymerase is not limiting, as it is during the plateau phase of the reaction. With appropriate standardization the procedure can be used for routine diagnostic purposes.

Protein-DNA conjugates produced by UV irradiation and their use as probes for hybridization.

Several proteins have been crosslinked to DNA by low dose uv irradiation. The principle of the method is based on an efficient and fast radiation induced reaction of amino acid residues with DNA at low pH. The method seems to be of general applicability for crosslinking proteins to DNA in a very simple one step procedure. Some of such DNA-protein conjugates have been used as probes for hybridization experiments. DNA-protein A probes were found to be most useful.

Gene analysis and the clinical chemist.

Knowledge of gene analysis methods and concepts will be important to the clinical chemist in the near future. Currently most gene analyses must be performed by indirect techniques, using polynucleotide probes hybridizing close to or on the disease gene but not on the position of the mostly unknown gene mutation (restriction fragment length polymorphism analysis). The sensitivity and specificity of such assays are affected by biological and methodologic factors, and are being continually improved. Preventive medicine is a promising area for gene analysis which will possibly fit well into the domain of clinical chemistry. The application of nucleotide hybridization analysis in tissue matching for organ transplantation, and in the detection and differential diagnosis of malignancies is in its early stages. A very promising, and rapidly emerging, technology is the direct detection and differentiation by gene probing of bacteria and viruses in medical microbiology. Guidelines for the ethical problems of gene analysis already exist within the field of medical ethics.

The cleavage of single-stranded DNA by the isoschizomeric restriction endonuclease HhaI and CfoI.

The cleavage of single-stranded (ss) M13mp8(+) DNA by the isoschizomeric restriction endonucleases HhaI and CfoI has been investigated. The two enzymes differ considerably in their ability to cleave ssDNA. HhaI cleaves ssDNA about two orders of magnitude faster than does CfoI, although both enzymes show the same activity when assayed on double-stranded DNA. From the cleavage of oligonucleotides and of M13mp8(+) DNA fragments it is concluded that cleavage of ssDNA occurs via transiently formed double-stranded hairpin structures. A rough correlation exists between the stability of the secondary structures and the cleavage efficiency.

Mutations induced by DNA polymerase alpha upon in vitro replication of M13mp8(+) DNA.

The forward mutation of the lacZ part of the bacteriophage M13mp8 has been used to study the fidelity of the 9S DNA polymerase alpha from calf thymus during in vitro replication of single-stranded DNA. Errors leading to a loss of alpha-complementation were identified by DNA sequencing. The overall mutation rate of the lacZ target sequence was in the range of 1:300-1:1000 which is more than one order of magnitude higher than the spontaneous mutation rate. In a mutL host the mutation rate was nearly threefold higher as compared to the wildtype host. Base substitutions comprise 86% of the errors whereas base deletions amount to 12%. The addition of a base was detected only in one mutant out of 71 sequenced ones. The frameshift mutations occurred predominantly in runs of the same base. The frequencies of individual base substitution are in the order of 2 X 10(-4)-4 X 10(-4) for most of the mismatches. Mutations involving dCTP:T and dGTP:T mismatches are observed with a lower frequency, those involving dTTP:C mismatches with a higher frequency.

Analysis of secondary structures in M13mp8 (+) single-stranded DNA by the pausing of DNA polymerase alpha.

The pausing of DNA replication has been used as a tool for analyzing secondary structures in a single-stranded DNA. M13mp8 (+) single-stranded DNA was replicated in vitro by the DNA polymerase alpha from calf thymus. The positions of pausing were determined from DNA sequencing gels. All experimentally observed pausing sites could be correlated with computer-predicted secondary structures of the M13 single-stranded DNA. In the computer calculations of the secondary structures, long-range base-pairing, G.T mispairs and loop-out of bases were allowed. By using six different primers, the pausing site pattern and the corresponding secondary structure map of a region comprising 1400 nucleotides of the M13 genome has been established. Our experiments indicate that the M13 DNA is highly structured. Most of the stable structures are clustered around the origin of replication. With fragments of the M13 DNA, we show that long-range base-pairing exists in the M13 single-stranded genome and we present evidence for tertiary structure interactions. Furthermore we observe structures that form newly during the course of replication. The Escherichia coli single-stranded DNA-binding protein facilitates replication through the barriers.

The elongation of mismatched primers by DNA polymerase alpha from calf thymus.

The ability of the 9S and 5.7S DNA polymerase alpha subspecies from calf thymus in elongating a mismatched primer terminus has been investigated. With poly(dA) as template, the elongation rate for (dT)8dG, (dT)8dC and (dT)10dGdT is 20-fold lower for the 9S enzyme and 5-fold lower for the 5.7S enzyme as compared to (dT)10. The presence of a second mismatch at the primer terminus reduces the elongation rate further by a factor of two. Exonucleolytic excision of the mismatches can be excluded. With (dT)8dG (dT)n as primer we show, that at least five T-residues have to follow the mismatch in order to establish the elongation rate of a perfectly paired primer. The KM value for (dT)10 dG as primer is 400 nM as compared to 10 nM for (dT)10. Addition of Mn2+ increases the relative efficiency of elongation of the mismatched primers.